1. Field of the Invention
This invention relates to and has among its objects novel compositions for therapeutic use and methods of making them. It is a particular object of this invention to provide compositions containing therapeutically active proteins which are substantially free from infectious agents such as viable viruses and bacteria, e.g. hepatitis viruses. Further objects of the invention will be evident from the following description wherein parts and percentages are by weight unless otherwise specified.
2. Description of the Prior Art
Many useful blood fractions and blood proteins are obtained from human blood plasma by fractionation according to known techniques such as, for example, the alcohol fractionation method of Cohn described in U.S. Pat. No. 2,390,074 (1945) and the Journal of the American Chemical Society, Vol. 68, page 459 (1946) and the Rivanol.RTM.-ammonium sulfate method. The aforementioned methods as well as other variations and techniques are summarized in "The Plasma Proteins", second edition, Volume III, pages 548-550, Academic Press, New York, N.Y. (1977). These blood fractions contain biologically active proteins that possess certain therapeutic qualities. For instance, Factor VIII or antihemophilic factor is useful against hemophilia; plasminogen is a precursor of plasmin for treatment of acute thromboembolic disorders; immune serum globulin (IgG) is employed in the treatment of congenital gamma globulin deficiency, and prophylaxis of measles, poliomyelitis and hepatitis A and B; fibronectin has been implicated as active in treatment of burns, shock, cancer, etc.; antithrombin III is a coagulation inhibitor; cryoprecipitate itself may be used directly for classical hemophilia; Plasma Protein Fraction (human) and albumin are useful in treatment of shock due to burns, crushing injuries, abdominal emergencies, and any other cause producing a predominant loss of plasma fluids and not red cells; immune globulin, intravenous is a substitute for immune serum globulin administerable in larger quantities; Anti-inhibitor coagulant complex, or Factor VIII Inhibitor Bypassing Activity (FEIBA) described in U.S. Pat. No. 4,160,025 as a blood-coagulation-promoting preparation for Factor VIII inhibitor patients; alpha-1-proteinase inhibitor (alpha-1-antitrypsin) can be employed in the treatment of emphysema; plasma growth hormone corrects pituitary growth deficiency, somatomedin is useful in correcting growth deficiencies, other immune serum globulins, e.g., IgA, IgD, IgE, and IgM, may be employed to treat various immune protein deficiencies; prealbumin (U.S. Pat. No. 4,046,877) is employed to increase immunologic competence; plasminogen-streptokinase complex (U.S. Pat. No. 4,178,368) can be administered to patients for treatment of thromboembolisms; ceruloplasmin, transferrin, haptoglobin, and prekallikrein have reagent and other uses.
One problem confronting users of plasma, plasma fractions, and compositions containing individual blood proteins is the chemical and thermal instability of the therapeutically active proteins contained therein. In many cases, substantial, and sometimes complete, losses of activity are observed if these proteins are mixed with certain chemicals or heated above physiological temperatures, i.e., above about 40.degree.-45.degree. C. Consequently, these items require special care during preparation and storage to minimize such deactivation.
The chemical and thermal instability of the aforementioned proteins renders them difficult to free from viral and bacterial components. Therapeutically active proteins isolated from plasma may contain bacterial agents and viruses, e.g., hepatitis virus, present in the source material for the protein fraction, namely, blood from a donor. A risk of contracting hepatitis exists, therefore, for those receiving fractions from blood plasma fractionation because the presence of the virus cannot be detected with certainty by any known procedure. In a large number of situations, this risk is outweighed by the detriment to a patient in not receiving the therapeutic plasma fraction as determined by the physician.
Some therapeutically active proteins derives from plasma have been pasteurized, i.e. heated to reduce hepatitis infectivity successfully. For example, it is well known that albumin can be pasteurized by heating at 60.degree. C. or 64.degree. C. for 10 hours (Gellis et al., J. Clin. Invest., Vol. 27, pages 239-244 (1948) in the presence of certain stabilizers such as N-acetyl-tryptophan and sodium caprylate. Individuals receiving this pasteurized material did not contract hepatitis, thus indicating the inactivation of hepatitis viruses while retaining the activity of albumin under the afore-described heating conditions. Plasma Protein Fraction (human) is also stabilized during pasteurization by the above method.
A process for pasteurizing the plasma protein plasminogen is disclosed by Baumgarten et al. in U.S. Pat. No. 3,227,626. An aqueous preparation containing 0.25-20 milligrams per milliliter (mg/ml) of plasminogen and further containing 0.1-0.5 molar lysine with a pH of 5.3-7.5 was heated at 60.degree. C. for 10 hours. As the patentee states, hepatitis virus was destroyed and the danger of transmitting hepatitis was removed with retention of plasminogen activity. Attempts to pasteurize plasminogen under the above conditions in the absence of lysine resulted in complete destruction of plasminogen activity. It is interesting to note that plasminogen cannot be stabilized with N-acetyl-tryptophan and sodium caprylate during pasteurization, nor can albumin and Plasma Protein Fraction (human) be pasteurized in the presence of lysine.
Singher has described a process for treating plasminogen to produce a material that is not contaminated with hepatitis virus (U.S. Pat. No. 2,897,123). In the patented pasteurization technique, aqueous solutions of plasminogen are heated at about 60.degree. C. for about 10 hours. The activity of plasminogen is retained if the solutions have a pH in the range not less than 3 nor greater then 6.5 and an ionic strength not greater than 0.3.
Another method for removing hepatitis virus from a biological material is described in U.S. Pat. No. 4,168,300. The material to be treated is contacted with a preparation, which may be agarose gel or beaded polyacrylamide plastic coupled with a variety of hydrophobic ligands. Plasma and albumin were subjected to the above purification technique to remove hepatitis virus.
Singher, in the aforementioned U.S. Pat. No. 2,897,123, lists some chemical and other types of methods of destroying hepatitis virus. The least effective of these methods involves the use of either nitrogen mustard or betapropiolactone. High energy irradiation in appropriate dosage is effective but destroys biological activity when applied to human blood products. Heat is recognized also as effective against hepatitis virus, the preferred treatment being heating the material at 60.degree. C. for 10 hours. Higher temperatures above 70.degree. C. for shorter intervals or lower temperatures for longer intervals have also been tried with successful results. However, it is important to note that higher temperatures are undesirable because of the potential for denaturation of the proteins. Furthermore, lower temperatures for long intervals are to be avoided because various proteolytic enzymes are activated under these conditions, and these activated enzymes cause protein degradation. Also, the use of temperatures lower than 60.degree. C. for pasteurization has not been shown consistently to yield a material that does not contain the infective virus.
As mentioned above, the recognition that heating at 60.degree. C. and 64.degree. C. for 10 hours successfully destroys the hepatitis viruses in albumin was made by Gellis et al., supra. Gellis et al. proved experimentally that albumin heated under the above conditions did not transmit hepatitis even if hepatitis virus was present prior to pasteurization. However, the authors noted that hepatitis virus survived heating at 56.degree. C. for one hour, a temperature usually employed for the inactivation of viruses. Thus, although heating at temperatures of about 56.degree. C. for one hour will deactivate most viruses, hepatitis virus is not inactivated; and materials containing hepatitis virus, which are heated at 56.degree. C. for one hour, cause infection of hepatitis in individuals receiving such materials.
Japanese Pat. No. 51-134878 (1976) teaches the stabilization of Factor XIII against heat inactivation (60.degree. C. for 10 hours) by using 10-20% (w/v) of a stabilizer such as a neutral amino acid, a monosaccharide, or a sugar alcohol.
Furthermore, in U.S. Pat. No. 4,297,344 there is disclosed a method of stabilizing coagulation Factors II, VIII, XIII, antithrombin III and plasminogen against heat in the presence of 1-3 molar amount of a certain amino acid and 20-60% (w/w) of a carbohydrate.
In the production of pharmaceutical preparations such as virus vaccines, methods or means are necessary to inactivate or at least attenuate, the virus. The means or methods, on the one hand, must destroy or substantially reduce the infectiousness, but, on the other hand, must preserve the antigenic characteristics. Customary inactivation agents include, for example, Formalin, betapropiolactone, ethyl ethylenimine (U.S. Pat. No. 3,636,196), toluidine blue with irradiation, hydroxylamine, ethylene oxide (U.S. Pat. No. 3,456,053), and lower alkyl esters of acetic acid (U.S. Pat. No. 3,655,871).
It is known that a 2:1 1,10-phenanthroline-cuprous ion complex is a potent reversible inhibitor of isolated E. coli DNA polymerase I (D'Aurora et al., Biochemical and Biophysical Research Communications, Vol. 78, No. 1, pages 170-177, 1977). This complex has also been shown to be an effective inhibitor of isolated E. coli DNA dependent RNA polymerase, isolated Micrococcus luteus DNA dependent DNA polymerase, and isolated T4 DNA dependent DNA polymerase (D'Aurora et al., ibid., Vol. 80, No. 4, pages 1025-1032, 1978).
Sigman et al (Journal of Biological Chemistry, Volume 254, No. 24, pages 12269-12272, 1979) demonstrated that under aerobic conditions the cuprous-phenanthroline complex catalyzes depolymerization of poly (dA-dT) and relaxation of closed supercoiled SV40 DNA. In vitro inhibition of polymerase activity was related to such strand scission of the primer/template.
It is important in the treatment of a proteineous composition such as for example, a plasma protein composition containing viral and bacterial components or a virus vaccine, that the viral and bacterial infectivity of the composition be substantially reduced or eliminated while at the same time retaining a high yield of a substantial portion of the activity of the proteins in the protein composition or the antigenic activity of the virus vaccine. Many prior art methods do not allow the user to obtain all of the above objectives.